The resistance of prostate cancer (PCa) to androgen deprivation therapy (ADT) and chemotherapy may be due to a subset of cancer cells characterized by low levels of prostate specific androgen (PSA) expression, scientists claim. Researchers at the University of Texas M.D. Anderson Cancer Center and colleagues have found that in contrast with prostate cancer cells that expressed high levels of PSA (PSA+ cells), PSA-lo cells expressed either no, or very low, levels of androgen receptor, making them resistant to hormonal therapy. Compared with the PSA+ subpopulation, PSA-lo cells also divided very slowly and expressed anti-stress genes and stem cell genes that enabled them to resist chemotherapy and retain long-term tumor-generating capacity.

PCa is known to contain differentiated cells that express high levels of PSA, as well as PCa cells that express little or no PSA. Moreover, prior research has indicated that PSA-lo cells are rare in early-stage tumors and become more abundant in high-grade and locally advanced tumors.

The M.D. Anderson Center researchers’ studies were designed to investigate whether the two subpopulations played different roles in tumor maintenance and progression to CRPC. To enable this they designed an approach to separating PSA-lo LNCaP cells from PSA+ LNCaP cells using a GFP-expressing lentiviral vector system and fluorescence-activated cell sorting (FACS).

Initial studies on the two lentiviral-vector expressing isogenic cell types indicated that in addition to their lack of androgen receptor protein expression, the PSA-lo cells demonstrated very different gene expression patterns compared with PSA+ cells. Up to 10% of the 561 genes significantly overexpressed in the PSA-lo cells were involved in antistress responses, including hypoxia response and DNA-damage sensing and repair. These cells also underexpressed many proapoptotic genes. In fact, when both the PSA-lo and PSA+ cell types were subjected to chemical stresses including etoposide, paclitaxel, or hydrogen peroxide, the PSA-lo cells expanded, while numbers of PSA+ cells decreased.

Interestingly, the PSA-lo LNCaP cells also underexpressed dozens of cell cycle and mitosis-related genes, indicating that they were a quiescent cell population, compared with the rapidly dividing PSA+ cells. This suggestion was supported by cell cycle analysis. Importantly, the PSA-lo LNCaP cell population preferentially expressed a number of stem cell and developmental genes, possessed a higher capacity to establish holoclones and anchorage-independent prostaspheres, and generated larger spheres and a greater number of secondary spheres than PSA+ cells.

The researchers moved on to carry out experiments using lentivector-expressing LAPC9 and LAPC4 xengraft tumors. PSA-lo cells from these tumors were, like the LNCaP cells, relatively quiescent. In suitable medium they generated spheres that expanded and could be passaged for at least four generations, whereas PSA+ LAPC9 cells generally aborted by the second generation, even though they formed larger primary spheres. The PSA-lo LAPC9 cells in addition expressed stem cell genes, were androgen-deprivation resistant, and possessed long-term tumor-propagating capacity when injected into hormonally intact male mice. In contrast, while PSA+ cells generated larger primary tumors in vivo, when tumors derived from each of the two cell types were serially passaged in mice, the PSA-/lo cells maintained relatively constant tumorigenicity, whereas PSA+. cells displayed decreasing tumorigenicity.

Importantly, when purified PSA+ and PSA-/lo LAPC9 cells were implanted in chemically fully castrated NOD/SCID mice, the low-PSA cells generated much larger, faster-growing tumors than PSA+ cells. Further genetic analysis and RNA inhibition studies showed that overexpression of Nanog, CD44 and OPN by these cells was necessary for the continued tumor-regenerating capacity of such cells.

Translating these findings to patient tumors, the investigators confirmed that low levels of tumor PSA mRNA correlated with reduced overall patient survival. Moreover, they found that PSA-lo cells separated from primary prostate tumor specimens were androgen receptor-negative and possessed much higher clonal and sphere-forming capacities than the corresponding PSA+ cells. Microarray analysis of four pairs of purified PSA-/lo and PSA+ HPCa cells showed that the PSA-lo cells expressed many stem cell and developmental genes. Serial transplants of reporter gene-expressing PSA-lo and PSA+ cells from human HPCa58 prostate cancer xenograft tumors into male NOD/SCID mice demonstrated that while the PSA+ cells initiated larger tumors in the first generation, on passaging, the PSA-lo HPCa58 cells developed larger tumors.

“One of the most significant contributions of the present work is to provide direct experimental evidence that PSA-/lo PCa cells may represent an important source of CRPC cells,” the authors conclude. “It is presently unclear how PSA-/lo and PSA+ cells, both of which are maintained under identical conditions, embark on different developmental fates. Nevertheless, the distinct division modes of PSA-/lo and PSA+ cells reinforce their intrinsic biological differences.”